1. Field of the Invention
The invention relates to a light calibration system consisting of a compact LED source with feedback control of intensity.
2. Brief Description of the Prior Art
Where photo- or video microscopy are employed as analytical techniques, a light standard providing preset levels of intensity over a homogeneous field covering the viewing area of a microscope is a useful accessory for system validation and testing. Functional studies of cell biolog routinely seek information about cellular activity by means of video imaging, using image sequences to monitor correlated cellular actions, such as calcium flux transients in relation to force generation in muscle cells. Quantitative information concerning intracellular ion concentration changes are obtained, in theory, using ratio imaging techniques, permitting degrees of cellular activity to be compared.
The reliability of photomicroscopic assays depends on accurate translation of light intensities, some of which are exceedingly faint, into pixel brightness levels. Unfortunately, video detection systems and accessories employed for low light recording cause distortions of both brightness and geometry in the image. Photoelectron multipliers in SIT and intensified SIT cameras exhibit nonlinear response at light levels below saturation. Electrostatically focused SIT cameras are subject to "shading" and "pincushioning". Pincushion distortion can vary over the range of intensification, while shading appears to vary slightly with average illumination in some cameras (unpublished data). These effects complicate the task of insuring the photometric accuracy of images obtained under different recording conditions. Newer solid-state microchannel plate intensifiers used with CCD cameras, which are free from shading and pin cushion effects, still are nonlinear and often impose onto the low light image a repeating pattern of varying intensity (chicken wire pattern) caused by the array of microchannels. The several forms of image distortion must be identified and corrected, or at least shown to be insignificant, before quantitative information can be obtained using photomicroscopy.
To aid in assessing the optical performance of photo- and video-microscopes as well as the reproducibility of recordings, standard light sources have been developed which are based on fluorescent microbeads, solid blocks and fluorescein filled glass capillary tubes. Beads permit the user to determine the degree to which the optical system faithfully images small three dimensional objects. Solid standards, such as uranyl glass and polymethacrylate blocks containing anthracene or rhodamine B (Starma Cells Inc.), undergo almost no photobleaching and are thus excellent tools to monitor fluctuations in excitation light levels. Solid standards have also been used to generate uniformly fluorescent fields from which to correct camera shading functions. Capillary tubes or cuvettes filled with known concentrations of fluorophores are used to characterize camera linearity, and can provide light intensities in absolute units with known excitation power, although in some cases the emission must be corrected for anisotropy. As useful as they are for quantitative work, chemical standards present some practical difficulties. The light output is subject to lamp noise from power fluctuations and aging of the burner. In addition, the solutions must be duplicated exactly to serve as calibration light sources, thus their preparation is both time consuming and difficult. Furthermore, most chemical standards show moderate photobleaching with use. Here we describe a solid-state light standard, employing feedback controlled light emitting diodes of differing wavelength, which does not have the disadvantages of chemical standards, and can serve both as a source of calibrated light and flat-field illumination.